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Synthesis of fluorescently labeled cerebrosides--N-[12-(9-anthryl)-11-trans-dodecenoyl]-1 beta-O-galactosylsphingosine and its 9-(3-perylenoyl)nonanoyl analog is described. Both probes are easily inserted in phosphatidylcholine vesicles.
During 4 months rats were given intraperitoneal injections of cerebrosides in a dose of 5 mg/kg bw. The measurements were taken of the arterial blood pressure and the function of blood cells. It has been demonstrated that as compared to the control, the experimental animals showed a significant elevation of the blood pressure, hyperaggregation of platelets and red cells, an increase in the ability of platelets to adhesion. It has been also disclosed that there was a decrease in the time of the occurrence of muscle fatigue and death under stress induced by forced dynamic work. The data obtained are of interest in the aspect of investigating the role played by glycolipids in the pathogenesis of cardiovascular diseases.
alpha-Cyclodextrin at concentrations of 1-8 mM helps dissolve, in aqueous solution, fatty acids such as lignoceric, stearic, and palmitic, and complex lipids such as ceramide and cerebroside that contain these acids. Formation of an inclusion complex was indicated on examination of the solution by gel filtration. alpha-Cyclodextrin strikingly increased synthesis of ceramide from sphingosine and either free lignoceric or stearic acid by rat brain preparations. These results suggest the further use of alpha-cyclodextrin in lipid enzymology, especially in relation to sphingolipid metabolism.
Suckling rats were exposed to methylmercury chloride or diethylmercury in order to induce chronic sublethal intoxication during the period of active myelination. Doses of 5 mg Hg/kg body weight were injected every second day from 5-25 days of age. The rats were killed at 27-28 days of age, and the brains contained about 1 microgram Hg/g wet weight. No changes in brain weight, myelin content of proteins or phospholipids were found, whereas the cholesterol and galactolipid levels were slightly reduced. The most significant change observed was a decrease in the ratio between alpha-hydroxy fatty acid and the nonsubstituted fatty acid in the myelin cerebrosides. The biochemical changes were less pronounced in the animals given diethylmercury than in animals receiving methylmercury.
An improved method for isolating cerebrosides from natural sources is described. The method is particularly suited to large scale work and can be adapted to the isolation of sphingolipids that are less polar than the gangliosides. It is based on the use of sodium sulfate to absorb the water from chloroform-methanol tissue extracts, the use of triiodide to cleave the ether linkage of plasmalogens, and the use of alkaline methanolysis to cleave the ester linkages of the glycerolipids. The final separation of the lipids is done with a silica gel column.
Cerebroside sulfotransferase (EC 2.8.2.11, CST) specific activity has been determined in oligodendrocyte (OL)-enriched glial cell cultures from newborn rat brain grown in serum supplemented medium. This activity is detectable at 5 days in vitro (DIV) and reaches its maximum value at 12 DIV. This period corresponds to that of oligodendrocyte precursor proliferation in these cultures. The activity decreases thereafter and remains nearly constant after 24 DIV. The developmental curve of CST activity is parallel in pure oligodendrocyte subcultures but twice higher than in primary cultures. These data confirm that CST is highly enriched in OL. Basic fibroblast growth factor (bFGF) (15 ng/ml) and platelet derived growth factor (PDGF) (0.75 U/ml) both enhance CST activity by 90% and 72%, respectively. This increase is in the same range than that of DNA content in treated cultures, whereas protein increase is smaller (50% and 22%, respectively). In contrast, transforming growth factor beta 1 (TGF beta 1, 0.5 and 5 ng/ml) does not significantly enhance CST activity nor DNA content of OL cultures. Insulin at high concentrations (5 micrograms/ml) also enhances CST activity but has no effect at physiological concentrations (20 ng/ml). These results show that CST activity can be controlled by growth factors. They suggest that CST activity is more closely related to OL and OL precursor proliferation than to myelination itself since its maximal activity preceeds myelination in vitro.
Previous results (Fressinaud, C., Sarliève, L.L., and Labourdette, G. J. J. Cell. Physiol., 141:667-674, 1989b) have shown that cerebroside sulfotransferase (CST; EC 2.8.2.11) is enriched in pure rat oligodendrocyte (OL) cultures and that its activity is increased by factors mitogenic for OL precursors and galactocerebroside (GC) expressing OL, such as basic fibroblast growth factor (bFGF), platelet-derived growth factor, and high insulin concentrations. In contrast, transforming growth factor beta or low insulin concentrations were found to be ineffective in this culture system. As bFGF mainly enhanced the proliferation of OL precursors (GC negative cells) rather than that of differentiated (GC+) cells, a relationship between OL precursor proliferation and CST increase was suggested. This hypothesis was first tested in 20-day-old OL cultures grown in chemically defined medium. The dose-response curve of [125I] Iododeoxyuridine ([125I]dUrd) incorporation toward bFGF was parallel to that of CST specific activity, and maximal stimulation was reached at 5 ng/ml bFGF for both. In contrast, 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP; EC 3.1.4.37) specific activity decreased after bFGF treatment. To determine if CST increase was linked to the proliferation of OL precursors induced by bFGF, cell proliferation was blocked by cytosine arabinoside (ARA-C). From 10(-8) to 10(-5) M ARA-C there was a dose-dependent inhibition of cell proliferation and a decrease in CST specific activity, whereas CNP specific activity was enhanced. When the cells were treated with bFGF and 10(-6) M ARA-C together, the proliferation was completely blocked and CST activity decreased by 72% below control values, whereas CNP activity was not significantly decreased. Immunocytochemical studies showed that the number of sulfatide-expressing cells and the number of cycling cells were increased after bFGF treatment, but that there was no overlapping between these two populations. Taken together these results suggest that CST activity and sulfatide expression appear shortly after the arrest of OL precursor division.
We have previously cloned the human UDP-galactose ceramide galactosyltransferase (CGT, E.C. 2.4.1.45) cDNA. Its open reading frame encodes the key enzyme in the biosynthesis of the glycosphingolipids, cerebrosides and sulfatides, essential constituents of the myelin membrane of the central nervous system (CNS) and PNS. Expression of the CGT gene and of the myelin-specific proteins in the terminal differentiated oligodendrocyte of CNS and in Schwann cells of PNS is cell-specific and highly time-regulated. The CGT gene therefore is important in the differentiation program of the oligodendrocyte lineage. Here we report the structural organization and the chromosomal localization of the human CGT gene. The coding sequence is separated into five exons, which are distributed over >40 kb. The CGT locus was mapped to the distal region of human chromosome 4, band q26. The organization of the CGT gene and of the UGT (uridylglucuronosyltransferases) gene family suggests a correlation to functional domains of the encoded proteins.
UDP-galactose ceramide galactosyltransferase, CGT, EC 2.4.1.45, is the key enzyme in the biosynthesis of cerebrosides and sulfatides, which are the most abundant glycosphingolipids in the myelin of the central nervous system and the peripheral nervous system. The cell-specific and highly time-regulated expression of the CGT gene is thought to play an important role in oligodendrocyte and Schwann cell differentiation. Three genomic clones encoding the mouse CGT gene were isolated and characterized. The gene is distributed over >42 kb, and the coding sequence is distributed over five exons ranging from 77 to 822 bp. Putative transcription start sites were determined by primer extension experiments. The CGT gene locus is highly conserved during evolution.
Cerebroside sulfate activator protein (CSAct or saposin B) is one of a group of heat stable, low-molecular-weight proteins that appear to share a common structural motif. These have been referred to as saposin-like proteins and are thought to share a multiple amphipathic helical barrel structure with a conserved pattern of disulfide linkages. Porcine kidney CSAct was prepared in high purity and consisted of three major glycosylated subforms. The protein was studied by physical-chemical methods and evaluated by various methods for structural prediction. All suggest that CSAct has high amounts of alpha-helical conformation and little if any beta-sheet. Circular dichroism (CD) studies indicate 45-50% helical conformation depending on buffer and temperature. There was only a moderate loss in helical content with increasing temperature and no indication of thermal denaturation. Fourier transform infrared spectroscopy (FTIR) measurements on deuterium hydrated self-films also indicated a predominantly helical structure. Helical axis orientation was investigated by both oriented CD and FTIR dichroism, which suggested that the helical axes were roughly parallel and oriented along the axis of the surface on which the self-films had been deposited. One-dimensional nuclear magnetic resonance spectra showed large chemical shift dispersion, indicating a defined tertiary structure with little variation between 6 and 85 degrees C. NOESY spectra failed to show the strong NOE cross peaks expected for a highly helical conformation. This may indicate short-term conformational flexibility within the helices or molecular aggregation at the high protein concentrations employed. These observations are consistent with the 3-4-helix bundle motif suggested for saposin-like proteins by various predictive algorithms.
The purification of cerebroside sulfate activator (CSAct) or saposin B from pooled human urine is described. Urinary proteins are concentrated by ammonium sulfate precipitation. A suspension of the precipitate is heat-treated and the heat-stable proteins are fractionated through a series of chromatographic steps. An initial concanavalin A column retains little of the CSAct activity, but is important for subsequent purification. Passing the Con A effluent directly onto an octyl Sepharose column removes the protein of interest which is recovered by affinity elution with octyl glucoside. Subsequent ion-exchange and gel filtration chromatographies yield a protein of 80-90% purity, although it is sometimes necessary to repeat one or more steps. A small amount of CSAct can sometimes be recovered from the initial Con A Sepharose column by methyl mannoside elution and purified by a parallel chromatographic protocol. Mass spectral analysis suggests that the final material is a mixture of two major and several minor glycoforms of a 79 amino acid protein with the structure predicted from the human prosaposin cDNA by truncation of both N- and C-terminal regions. Sugar analysis revealed the presence of glucosamine, mannose, and fucose, consistent with the major isoforms bearing a five-sugar Man(2)GluNac(2)Fuc or a single GluNac substituent. The human urinary material is similar to the previously characterized pig kidney protein in most respects, but varies in some details.
Cerebroside sulphate or sulphatides, present in erythrocyte and leucocyte cell membranes, have recently been shown to be a potent activator of the contact phase of coagulation. Platelet factor-four (PF4), purified from human washed platelets, was found to inhibit the sulphatide induced APTT and plasma prekallikrein activation in a dose dependent manner. A four-fold increase in the sulphatide-APTT was observed in 156pM PF4 in the reaction mixture and 30% inhibition of prekallikrein activation was observed at 1.6nM. PF4 was found to be an approximately 1000-fold more potent inhibitor than protamine sulphate in inhibiting activation of clotting but was not as potent in inhibiting activation of prekallikrein. The physiological form of PF4 as released from platelets, was also found to cause inhibition of plasma prekallikrein activation, with 50% inhibition observed when the concentration of PF4 in plasma was 2.25 g/ml (0.29 M). The ability of PF4 to inhibit the contact activation reactions induced by sulphatides may be of some physiological significance in that when released during platelet activation and vessel wall injury, it may have a protective effect against the development of thrombosis.
Previous studies on experimental allergic encephalomyelitis have shown that a number of leukocytes appear in demyelinating lesions of guinea-pig brain. The present studies showed that cerebroside sulfuric ester (sulfatide), a typical component of myelin membranes, stimulated the oxidative metabolism of guinea-pig neutrophils and macrophages, leading to marked generation of oxygen radicals and light emission. Formation of a spin adduct of 5,5-dimethyl-1-pyrroline N-oxide by leukocytes was dependent on the concentration of sulfatide, and correlated well with the generation of superoxide anion and the intensity of chemiluminescence measured in the absence of luminol. The addition of myelin membranes to the sulfatide-stimulated neutrophils amplified the light emission, suggesting an interaction between myelin membranes and those of leukocytes. Assay of the thiobarbituric acid reaction in the mixture of membranes and cells showed that sulfatide-stimulated cells induced lipid peroxidation in myelin membranes. These results suggest that sulfatide released from demyelinating lesions stimulates leukocytes to release toxic oxygen radicals, which attack myelin membranes, leading to a chain reaction of demyelination.
A highly convergent synthetic approach was developed to obtain alpha-galactosyl cerebroside O-(alpha-D-galactopyranosyl)-2-hexacosylamino-D-ribo-1,3,4-octa decantriol, which has previously been demonstrated to have immunostimulatory activity. Known 4,6-O-benzylidene galactose was the starting material for both the required alpha-galactosyl and the phytosphingosine building blocks O-(2,3-di-O-benzyl-4,6-O-benzylidene-D-galactopyranosyl) trichloroacetimidate (4) and 2-O-methanesulfonyl-D-arabino-1,2,3,4-octadecantetrol (5). The key step of the synthetic strategy is the highly regio- and stereoselective O-galactosylation of 1,3,4-O-unprotected phytosphingosine acceptor 5 using known 4 as donor. The total synthesis required only 11 synthetic steps starting from galactose.
The total synthesis of D-erythro-sphingosine (9) was performed by a chirospecific method starting from D-galactose via an azidosphingosine intermediate to give highly homogeneous (>99.9% C18:1) sphingosine base (9) which contained no observable olefin isomerization by product and was demonstrated to be optically pure by a novel method utilizing Mosher's acid. Ceramide (10) was prepared from this sphingosine (9) with highly homogeneous (99.8% C16:0) palmitic acid by two methods. The cerebroside glucosylceramide (23) was the next sphingolipid in this series to be synthesized in a highly homogeneous form. These three sphingolipids are currently being used for biophysical studies of the structures of their hydrated bio-molecular assemblies.
Sulfatide is a major acidic glycolipid in human gastric mucosa, and its sulfation is catalyzed by cerebroside sulfotransferase (CST). To investigate the expression of the CST gene in human gastric cancer, a reverse transcription PCR method was developed with the use of endoscopic bioptic specimens. By this method, we examined the CST mRNA expression in 11 cases of gastric cancer, and in all the cases we detected various levels of the expression both in cancer tissues and in uninvolved adjacent tissues. The present assay method was suggested to be useful in the detection of CST mRNA from a limited amount of bioptic samples.
Cerebroside sulfate activator (CS-Act) is a small compact protein which binds and solubilizes certain glycosphingolipids. Following the recent publication of the purification and preliminary sequence of pig kidney CS-Act [Fluharty, A.L., Katona, Z., Meek, W.E., Frei, K., & Fowler, A.V. (1992) Biochem. Med. Metab. Biol. 47, 66-85], we now report the primary sequence of the C-terminal portion of this protein and the assignment of the three disulfide bonds. Cyanogen bromide (CNBr) treatment of native CS-Act produced three major and several minor peptide fragments. Analysis of one HPLC-purified fragment revealed the C-terminus 14 amino acid sequence. This established the length of the native protein at 79 residues. In conjunction with the sequence data for one other major HPLC-purified CNBr fragment, it could be concluded that the three intrachain disulfide bonds were located at half-cystine residues 4 and 77, 7 and 71, and 36 and 47. Mass spectrometry (fast atom bombardment and electrospray ionization) showed the molecular weight of the major component of the CS-Act preparation to be 9720.5 Da, which was in close agreement with the calculated mass of the 79 amino acid peptide with five covalently attached sugar residues and three internal disulfide bonds. The mass spectrometric molecular weight measurements also showed that the CS-Act preparation possessed microheterogeneity in its carbohydrate moiety, as less intense signals corresponded to species containing (in decreasing order of abundance) two, one, four, and three sugar residues.(ABSTRACT TRUNCATED AT 250 WORDS)
Raman spectroscopy was used to study the phase behavior of several semisynthetic species of the acidic glycosphingolipid cerebroside sulfate (CBS) which occur in myelin. The C-H stretching mode region at 2800-3100 cm-1 of C18:0-CBS, C24:0-CBS, and C26:0-CBS, and the alpha-hydroxy fatty acid species C18:0h-CBS, was studied in the presence of 2 M Li+ and 2 M K+. Earlier studies have shown that K+ shields the negative charge on the sulfate more effectively than Li+, thus promoting intermolecular hydrogen-bonding interactions between the lipid molecules. Indeed, a novel broad background feature was present in the Raman spectra from 2900 to 3200 cm-1, which was attributed to O-H stretch associated with intermolecular hydrogen bonding between lipid hydroxyl groups. After subtraction of this broad feature, the intensities of the lipid C-H stretching vibrational transitions could be determined. These indicated that in K+, the degree of order (intrachain conformation and lateral chain-chain interactions) of C18:0-CBS, whose hydrocarbon region is fairly symmetrical in chain length, is similar to that of the symmetric chain length glycerolipid dipalmitoylphosphatidylcholine, while the degree of order is lower in Li+, as a result of the increased lateral charge repulsion of the head groups in Li+. Two phase transitions were observed for the highly asymmetric species C24:0-CBS and C26:0-CBS in K+ but only one transition in Li+.(ABSTRACT TRUNCATED AT 250 WORDS)